ABSTRACT OBJECTIVE:The link between maternal under-nutrition and cardiovascular disease (CVD) in the offspring later in life is well recognized, but the impact of maternal over-nutrition on the offspring's cardiovascular function and subsequent risk for CVD later in life remains unclear. Here, we investigated the impact of maternal exposure to a high-fat/calorie diet (HFD) during pregnancy and early postnatal period on endothelial function of the offspring in a nonhuman primate model.METHODS:Offspring, naturally born to either a control (CTR) diet (14% fat calories) or a HFD (36% fat calories) consumption dam, were breast-fed until weaning at about 8 months of age. After weaning, the offspring were either maintained on the same diet (CTR/CTR, HFD/HFD), or underwent a diet switch (CTR/HFD, HFD/CTR). Blood samples and arterial tissues were collected at necropsy when the animals were about 13 months of age.RESULTS:HFD/HFD juveniles displayed an increased plasma insulin level and glucose-stimulated insulin secretion in comparison with CTR/CTR. In abdominal aorta, but not the renal artery, acetylcholine-induced vasorelaxation was decreased remarkably for HFD/HFD juveniles compared with CTR/CTR. HFD/HFD animals also showed a thicker intima wall and an abnormal vascular-morphology, concurrent with elevated expression levels of several markers related to vascular inflammation and fibrinolytic function. Diet-switching animals (HFD/CTR and CTR/HFD) displayed modest damage on the abdominal vessel.CONCLUSION:Our data indicate that maternal HFD exposure impairs offspring's endothelial function. Both early programming events and postweaning diet contribute to the abnormalities that could be reversed partially by diet intervention.International Journal of Obesity advance online publication, 27 March 2012; doi:10.1038/ijo.2012.42.

[Show abstract][Hide abstract]ABSTRACT:
Background
The development of long-term vascular disease can be linked to the intrauterine environment, and maternal nutrition during gestation plays a critical role in the future vascular health of offspring. The purpose of this investigation was to test the hypothesis that a high-energy (HE) gestational diet, HE post-weaning diet, or their combination will lead to endothelial dysfunction in offspring.Methods
Duroc x Landrace gilts (n¿=¿16) were assigned to either a HE (10,144 Kcal/day, n¿=¿8) or normal energy (NE: 6721 Kcal/day, n¿=¿8) diet throughout pregnancy. Piglets were placed on either a NE or HE diet during the growth phase. At 3 months of age femoral arteries were harvested from offspring (n¿=¿47). Endothelial-dependent and -independent vasorelaxation was measured utilizing wire-myography and increasing concentrations of bradykinin (BK) and sodium nitroprusside (SNP), respectively.ResultsBK and SNP induced vasorelaxation were significantly reduced in the femoral arteries of gestational HE offspring. However, no effect for the post-weaning diet on BK and SNP induced vasorelaxation was seen. This investigation demonstrates that a HE diet prenatally diminishes both BK and SNP induced vasorelaxation in swine.Conclusions
These findings suggest that a HE gestational diet can play a critical role in the development of offspring¿s vascular function, predisposing them to endothelial dysfunction. This dysfunction may lead to atherosclerotic disease development later in life.

[Show abstract][Hide abstract]ABSTRACT:
Extensive experimental animal studies and epidemiological observations have shown that environmental influences during early development affect the risk of later pathophysiological processes associated with chronic, especially noncommunicable, disease (NCD). This field is recognized as the developmental origins of health and disease (DOHaD). We discuss the extent to which DOHaD represents the result of the physiological processes of developmental plasticity, which may have potential adverse consequences in terms of NCD risk later, or whether it is the manifestation of pathophysiological processes acting in early life but only becoming apparent as disease later. We argue that the evidence suggests the former, through the operation of conditioning processes induced across the normal range of developmental environments, and we summarize current knowledge of the physiological processes involved. The adaptive pathway to later risk accords with current concepts in evolutionary developmental biology, especially those concerning parental effects. Outside the normal range, effects on development can result in nonadaptive processes, and we review their underlying mechanisms and consequences. New concepts concerning the underlying epigenetic and other mechanisms involved in both disruptive and nondisruptive pathways to disease are reviewed, including the evidence for transgenerational passage of risk from both maternal and paternal lines. These concepts have wider implications for understanding the causes and possible prevention of NCDs such as type 2 diabetes and cardiovascular disease, for broader social policy and for the increasing attention paid in public health to the lifecourse approach to NCD prevention.

[Show abstract][Hide abstract]ABSTRACT:
Maternal high fat intake during pregnancy and lactation can result in obesity and adverse cardio-metabolic status in offspring independent of postnatal diet. While it is clear that maternal high fat intake can cause hypertension in adult offspring, there is little evidence regarding the role of dietary interventions in terms of reversing these adverse effects. Conjugated linoleic acid (CLA) is an omega 6 fatty acid with beneficial effects in obesity and metabolic status. However, the impact of CLA supplementation in the context of pregnancy disorders and high fat diet-induced developmental programming of offspring cardio-metabolic dysfunction has not been investigated. We have utilised a model of maternal overnutrition to examine the effects of CLA supplementation on programmed endothelial dysfunction during adulthood. Female Sprague-Dawley rats were fed either a purified control diet (CON) or purified control diet supplemented with 1% CLA (of total fat), a purified high fat (HF) diet (45%kcal from fat) and a purified HF diet supplemented with 1% CLA (of total fat) (HFCLA). All dams were fed ad libitum throughout pregnancy and lactation. Offspring were fed a standard chow diet from weaning (day 21) until the end of the study (day 150). Systolic blood pressure (SBP) was measured at day 85 and 130 by tail cuff plethysmography. At day 150, offspring mesenteric vessels were mounted on a pressure myograph and vascular responses to agonist-induced constriction and endothelium-dependent vasodilators were investigated. SBP was increased at day 85 and 130 in HF and HFCLA adult male offspring compared to CON and CLA groups with no effect of CLA supplementation. An overall effect of a maternal HF diet was observed in adult male vessels with a reduced vasoconstrictor response to phenylephrine and blunted vasodilatory response to acetylcholine (ACh). Furthermore, HF and HFCLA offspring displayed a reduction in nitric oxide pathway function and an increased compensatory EDHF function when compared to CON and CLA groups. These data suggest that a maternal HF diet causes a developmental programming of endothelial dysfunction and hypertension in male offspring which can be partially improved by maternal CLA supplementation, independent of offspring body weight.

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ORIGINAL ARTICLEMaternal high-fat diet impacts endothelial function innonhuman primate offspringL Fan1,2, SR Lindsley1, SM Comstock1, DL Takahashi1, AE Evans1, G-W He2, KL Thornburg3and KL Grove1OBJECTIVE: The link between maternal under-nutrition and cardiovascular disease (CVD) in the offspring later in life is wellrecognized, but the impact of maternal over-nutrition on the offspring’s cardiovascular function and subsequent risk for CVDlater in life remains unclear. Here, we investigated the impact of maternal exposure to a high-fat/calorie diet (HFD) duringpregnancy and early postnatal period on endothelial function of the offspring in a nonhuman primate model.METHODS: Offspring, naturally born to either a control (CTR) diet (14% fat calories) or a HFD (36% fat calories) consumptiondam, were breast-fed until weaning at about 8 months of age. After weaning, the offspring were either maintained on thesame diet (CTR/CTR, HFD/HFD), or underwent a diet switch (CTR/HFD, HFD/CTR). Blood samples and arterial tissues werecollected at necropsy when the animals were about 13 months of age.RESULTS: HFD/HFD juveniles displayed an increased plasma insulin level and glucose-stimulated insulin secretion incomparison with CTR/CTR. In abdominal aorta, but not the renal artery, acetylcholine-induced vasorelaxation was decreasedremarkably for HFD/HFD juveniles compared with CTR/CTR. HFD/HFD animals also showed a thicker intima wall and anabnormal vascular-morphology, concurrent with elevated expression levels of several markers related to vascular inflammationand fibrinolytic function. Diet-switching animals (HFD/CTR and CTR/HFD) displayed modest damage on the abdominal vessel.CONCLUSION: Our data indicate that maternal HFD exposure impairs offspring’s endothelial function. Both early programmingevents and postweaning diet contribute to the abnormalities that could be reversed partially by diet intervention.International Journal of Obesity (2013) 37, 254--262; doi:10.1038/ijo.2012.42; published online 27 March 2012Keywords: over-nutrition; programming; intrauterine; endothelium; juvenile offspringINTRODUCTIONThe worldwide prevalence of childhood obesity has increaseddramatically in the past several decades.1Numerous epidemiolo-gical studies have documented a close association betweenearly-onset obesity and the risk for developing cardiovasculardiseases (CVDs) in adulthood.2--4Maternal health and diet duringpregnancy and the early postnatal period is now known to be themajorcontributing factor tobolic disorders in obese children.5--7A clear association hasbeen established in humans between nutritional restriction andplacental insufficiency during pregnancy with the connection toCVD in their offspring later in life. These effects have beenconfirmed in sheep models.8--12In addition, there are data inrodent models,13--15demonstrating that maternal over-nutritioncan impair cardiovascular function in obese offspring as adults;however, it was not determined if the endothelial dysfunctionoccurred before the onset of obesity. Our current study aims todetermine the relative impact of exposure to a high-fat/caloriediet(HFD)duringtheprenatalon endothelial function in juvenile nonhuman primate (NHP)offspring.We have established a maternal HFD NHP model,16wheremonkeys are maintained on a HFD before and throughoutpregnancy and lactation. Using this model, our ongoingstudies16--21demonstrated that consumption of a HFD duringpregnancy leads to broad developmental health issues in thethemanifestation ofmeta-and/orpostnatalperiodoffspring, independent of maternal obesity and insulin resistance.These studies have found that the fetal offspring demonstratedrestricted fetal growth, placental insufficiency accompanied by adecrease in placental blood flow volume, increased release ofcytokines, dyslipidemia and an increased deposition of fat in theliver. Moreover, after birth, the HFD offspring displayed catch-upgrowth, increased fat mass and persistent fatty liver. These are allhallmarks for an increased risk of CVD. However, few studies todate have been conducted on the effects of maternal HFD intakeon the cardiovascular system of the offspring, especially on anNHP model.High-fat dietary intake is a major risk factor for the developmentof obesity. Obesity is closely associated with a number ofestablished cardiovascular risk factors, including diabetes mellitus,insulin resistance, dyslipidemia and hypertension, which arecumulatively damaging to endothelial function.22--24The structur-al and functional integrity of the vascular endothelium has acrucial role in cardiovascular homeostasis. Abnormal endothelialfunction is a well-recognized precursor of atherosclerotic disease.Under normal circumstances, the vascular endothelium releasesvarious vasodilator and vasoconstrictor substances that regulatelocal vascular tone to ensure adequate blood flow, as well asregulate platelet aggregation and leukocyte adhesion to theendothelium. All of these effectively counteract the onset ofatherosclerosis.25On the contrary, in a state of insulin resistance,such as obesity and diabetes, endothelium-dependent vasodilatorReceived 8 September 2011; revised 24 January 2012; accepted 25 February 2012; published online 27 March 20121Division of Neuroscience, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR, USA;2Cardiovascular Research Laboratory, StarrAcademic Center, Providence Heart and Vascular Institute, Portland, OR, USA andCorrespondence: Dr KL Grove, Division of Neuroscience, Oregon National Primate Research Center, Oregon Health and Science University, 505 NW 185th Avenue, Beaverton,OR 97006, USA.E-mail: grovek@ohsu.edu3Heart Research Center, Oregon Health and Science University, Portland, OR, USA.International Journal of Obesity (2013) 37, 254--262& 2013 Macmillan Publishers LimitedAll rights reserved 0307-0565/13www.nature.com/ijo

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response,function,26is depressed.27--30Based on the evidence that endothelial dysfunction is thecentral pathogenicfeatureofcardiovascular disorders,30--32and based on our previous findings,we hypothesized that maternal HFD consumption during preg-nancy and the perinatal period would impair the endothelialfunction of the offspring. In order to test our hypothesis, wecollected abdominal aorta and renal arteries from 13-month-oldNHP juvenile animals to test endothelial function, using multipletechniques. Apart from understanding the programming effectson the CVD risk in the juvenile offspring as a consequenceof maternal obesity, this study also aimed to determine thecontribution of dietary vs early programming events to endothe-lial dysfunction. In addition, these studies investigate the possiblebeneficial contribution of dietary intervention in juvenile animals,after the potential early programming events have occurred.the most useful measurement ofendotheliummetabolic-disease-associatedMATERIALS AND METHODSNHP modelAll animal procedures were in accordance with the guidelines of theInstitutional Animal Care and Use Committee of the ONPRC (OregonNational Primate Research Center) and conformed to National Institutes ofHealth guidelines on the ethical use of animals. The completecharacterization of the maternal and fetal phenotype has been describedin our previous studies.22--27AdultsYoung adult female Japanese macaques (Macaca fuscata) were age-(5--7 years old) and weight-matched (7--9kg) and maintained on a control(CTR) or a HFD up to 5 years. The CTR diet contained 14% calories from fat(Lab Diet, 5052, Purina Mills Inc., St Louis, MO, USA) supplemented withfruits and vegetables, and the HFD supplied 36% of calories from fat(Test Diet, 5L0P, Purina Mills Inc.) supplemented with calorically densetreats. The composition of this HFD represents a typical Western diet inregards to the saturated fat content. All animals were socially housedin indoor/outdoor pens (male-to-female ratio of 1--2:5--9) and hadad libitum access to food and water.13-Month-old juvenilesOffspring, naturally born to either a CTR or a HFD consumption dam, weremaintained with their dams until about 8 months of age (249±4 days).After weaning, some offspring were maintained on the same diet as theirmoms, fed either a CTR diet (group 1: CTR/CTR, denoted as C/C) or a HFD(group 2: HFD/HFD, denoted as H/H); others underwent a diet switch fromeither HFD to CTR (group 3: HFD/CTR, denoted as H/C) or from CTR to HFD(group 4: CTR/HFD, denoted as C/H). All the juveniles were socially housed.At B13 months of age (393±4 days), all juveniles from the four treatmentgroups were sent to necropsy for blood and tissue collection. The datacollected from these animals were from three different cohorts of animalsover 3 years and were balanced for offspring sex and maternal dietsensitivity.Measurements of metabolic phenotypesBefore necropsy, all the juvenile offspring underwent a morning fast. In theafternoon, the animals were deeply anesthetized with Nembutal and thebody weights were measured. Blood samples were collected for the assess-ment of their metabolic status, including blood glucose level (determinedby One-Touch Glucometer (LifeScan, Inc., Milpitas, CA, USA), which demon-strated a good analytical performance with a bias o10% from the comparativeclinic and laboratory methods in humans33), plasma insulin and triglycerideslevels, and serum free fatty acids level (assayed by using the correspondingkit). Homeostatic model assessment of insulin resistance (HOMA-IR) wascalculated by the formula glucose (mgdl--1)?insulin (mUml--1)/405.Area under the curve for glucose (GAUC) and insulin secretion (IAUC)were calculated based on an ivGTT test (intravenous glucose tolerancetest), which was conducted for each animal 1 week before it was sent tonecropsy. In detail, after an overnight fast, the animal was then sedatedwith Telazol (3mgkg--1) and a baseline blood sample was collected. Afteran intravenous administration of a high dose of glucose (0.6gkg--1ofsterile 50% dextrose solution), a series of blood samples were collected at1, 3, 5, 10, 20, 30, 45 and 60min thereafter. GAUC and IAUC were thencalculated based on the glucose and insulin concentrations at each timepoint. In addition, total body fat for each animal was determinedconcurrently with the ivGTT test by conducting DEXA scans (HologicDiscovery A, Hologic Inc., Bedford, MA, USA) during the sedation period.Preparation of abdominal aorta and renal artery tissueAt necropsy, the far end of the descending abdominal aorta rings and therenal arteries were removed and dissected, with some of the tissuebeing snap frozen, and some submersed in 4% paraformaldehyde(pH¼7.4) or neutral formalin.Organ bath technique and the recording of acetylcholine-inducedvasorelaxationFreshly collected abdominal and renal arterial rings, in 3-mm lengths, weremounted in an organ bath, followed by a 60-min resting period.34,35Therings were then stretched in progressive steps to reach their optimallumen pressure. After equilibration for another 60min, the rings werepre-contracted by U46619 (9,11-dideoxy-11a, 9a-epoxy-methanoprosta-glandin F2a; Cayman Chemical Company, Philadelphia, PA, USA; 10nM).When the contraction reached a stable plateau, cumulative doses ofacetylcholine (ACh; Sigma Chemical Co., St Louis, MO, USA; ?10B ?5.5LogM) were added by 0.5-Log steps to produce a vasorelaxant response.The cumulative concentration-relaxation curves were then recorded. Thevasorelaxant response to ACh at each dose point for each ring was finallyexpressed as a percentage decrease from U46619-induced pre-contraction.EC50value for each curve was also calculated.Kreb’s solution of the following composition (mM): Naþ144, Kþ5.9,Ca2þ2.5, Mg2þ1.2, Cl?128.7, HCO3aerated with a gas mixture of 95% O2and 5% CO2at 37±0.11C wasutilized throughout the experiments.?25, SO42?1.2, H2PO4?1.2, Glucose 11,Quantitative real-time polymerase chain reaction and geneexpression level determinationThe total RNA of the arterial tissue was isolated. Then, 1mg RNA of eachsample was prepared for real-time reaction and the mRNA expression levelfor each gene was measured by quantitative real-time polymerase chainreaction using a PRISM 7700 Sequences Detector System (Applied Biosystems,Foster City, CA, USA). The monkey 18s gene was used as an internal marker.The gene expression level was calculated by its individual standard curve,and then normalized to 18s level for each sample. The primers and probesequences (50--30), designed against macaque mRNA for all the target genes,were purchased from Applied Biosystems and Invitrogen (Carlsbad, CA, USA),including endothelial nitric oxide synthase (eNOS) (Hs00167166_ml), plasmi-nogen activator inhibitor-1 (PAI-1) (Hs01126607_g1), tissue-type plasminogenactivator (t-PA) (For: TGACTGCTACCTTGGGAATGG; Rev: TGCCTATCAGCATCATGGAATT; Probe: CACAGCCTCACCGCATCGGGT), VCAM-1 (vascular cell adhesionmolecule-1) (For: TGAGAGGCAGACTTCCCTGAAT; Rev: TTTTCAGGCAGCAAGTTTTCC; Probe: CACCTATGCCCCTTGCTCTGAGCAAG), intercellular adhesionmolecule-1 (ICAM-1) (For: TAGCCGGCCAGCTTGTACAC; Rev: TGGCCACGTCCAGTTTCC; Probe: TCCGTGTCCTGTATGGACCCCGA), vascular endothelial growthfactor (VEGF) (For: GAAGTGGTGAAGTTCATGGATGTC; Rev: CACCAGGGTCTCGATTGGAT; Probe: ATCAGCGCAGCTACT), tumor necrosis factor a (TNFa)(For:CTCTGGCTCAGGCAGTCAGAT;Probe: ATCTTCTCGAACCCC), MCP-1 (monocyte chemotactic protein-1)(For: AGGCTGGCGAGCTATAGAAGAAT; Rev: TCTTGAAGATCACAGCTTCTTTGG; Probe: ACCAGCAGCAAGTGT) and IL-6 (interleukin 6) (For: CAGATATGAACTCCGTCTCCACAA; Rev: AGCAGCCCCAGGGAGAAG; Probe: CGCCTTCGGTCCAGTT).Rev:CATGGGCTACAGGCTTGTCA,Programming of endothelial dysfunctionL Fan et al255International Journal of Obesity (2013) 254--262& 2013 Macmillan Publishers Limited

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Hematoxylin/eosin staining for morphological assessmentH&E (hematoxylin/eosin)-stained paraffin-sliced sections of abdominalaorta tissue from C/C and H/H animals were used for the morphologicalassessment, specifically the changes underneath the endothelium mono-layer. Pictures were then taken using Slidebook5.0 (Intelligent ImagingInnovations, Inc., Denver, CO, USA).Immunohistochemistry for measurement of intima thicknessParaffin-sliced sections containing abdominal aorta rings were rehydrated,followed by antigen retrieval. Then, sheep polyclonal anti-CD31primaryantibody (1:400, #AF806, R&D Systems Inc., Minneapolis, MN, USA) wasapplied after blocking and left overnight. The next day, sections wereincubated with the secondary antibody, followed by A/B solution andNickel DAB solution treatment. Finally, the sections were covered with astrip of Permount and left to dry for photographing. Intima thickness of theabdominal aorta rings was then measured as the distance between theendothelium monolayer and the elastic tissue underneath, using theLeica Application Suite (version 3.5.0 software, Buffalo Grove, IL, USA).Three rings from each aorta were analyzed with eight equally spacedmeasurements. Therefore, 24 total measurements yielded an average valuefor each animal, which was used for the final calculations and statisticalcomparisons.Statistical analysisData were expressed as mean±s.e. Statistical comparisons of thecumulative percentage of relaxation under different treatments wereperformed by two-way analysis of variance, followed by Bonferroni’s test.EC50value, gene expression level and intima thickness comparisons forvarious groups of animals were performed by one-way analysis of variance,followed by Bonferroni’s multiple comparison test analysis. Statisticalcomparisons between two groups were performed by t-tests. Correlativerelationships were analyzed by Pearson’s correlation analysis. A P-valueo0.05 was considered statistically significant.RESULTSMetabolic phenotypeAt the time of necropsy (13 months of age), there was nodifference in body weight between groups (Table 1). The totalbody fat was o3% for all animals. Despite the lean phenotype,specifically in H/H offspring, there was an elevation in plasmafasting insulin level, as well as glucose-stimulated insulin secretion(IAUC) and HOMA-IR in comparison with C/C, indicating that H/Hjuveniles were developing insulin resistance. However, fastingglucose values were normal in these offspring. There was also nodifference between groups in fasting free fatty acids andtriglycerides. Importantly, the animals exposed to the HFD onlyduring the postweaning period (C/H) and the postweaning dietintervention group (H/C) displayed intermediate values for fastinginsulin, IAUC and HOMA-IR, that were not significantly differentfrom either the C/C or H/H groups.ACh-induced endothelium-dependent vasorelaxation responseThe measurement ofACh-inducedvasorelaxation response is the most useful test of endothelialfunction. Thus, in this study, we investigated the effect of maternaland early postnatal HFD exposure on endothelium-dependentvasorelaxation of the offspring by performing ACh-inducedconcentration-relaxation response on both abdominal aorta andrenal artery rings.As shown in Figure 1a, the maximal vasorelaxation response(Rmax) produced by ACh was remarkably decreased in abdominalaorta rings taken from H/H juveniles (44.6±6.2%, n¼10)compared with C/C (79.0±7.8%, n¼9), along with a 4300%higher EC50 value for the corresponding relaxation curve(?7.102±0.143 vs ?7.634±0.103 LogM, Po0.01; Figure 1b). Onthe contrary, C/H juveniles that have a similar adiposity,demonstratedanunchangedwhereas the H/C animals completely reversed the depressedvasodilatation. Concurrently, the EC50value against the curve forH/C animals was 11-fold lower in comparison with H/H juveniles(?8.170±0.280 LogM vs ?7.102±0.143). Furthermore, the vesselsof H/C animals tended to be more sensitive in response to ACh,with a slightly left-shifted curve in comparison with C/C. Never-theless, EC50values against the ACh-induced vasorelaxant curvefor juveniles exposed to the maternal HFD (H/H and H/C) weredemonstrated to be positively correlated with IAUC (r¼0.53,Po0.05; Figure 1c). These data suggest that maternal HFDexposure contributes to the detrimental effect of insulin resistanceon vasorelaxant function in offspring.Noteworthy, all of the tested abdominal aorta rings were undersimilar pre-contraction forces induced by thromboxane A2analogU46619before thevasorelaxation(Figure 1d).Interestingly, there was no difference in renal artery endo-thelial responses (83.4±8.6%, 82.1±7.4%, 77.0±11.2% and57.3±6.4%, n¼4--9; Supplementary Figure S1) between any ofthe groups.endothelium-dependentvascularrelaxationresponse,responseswereelicitedeNOS gene expressioneNOS is of critical importance in ACh-induced endothelium-dependent vasodilatation. In order to determine whetherthe depressed ACh-induced vasorelaxation response is due tothe depressed expression of eNOS in the vessel, we measured theeNOS mRNA level in abdominal aorta tissue from various groupsof juvenile offspring, using quantitative real-time polymeraseTable 1.Metabolic phenotype for the Juvenile offspringC/CH/HH/C C/HNBody weight (g)Total body fat (%)Fasting glucose (mgdl--1)Fasting insulin (mUml--1)HOMA-IRAUC for insulin (IAUC)AUC for glucose (GAUC)Free fatty acids (FFA; mEgl--1)True triglycerides (mgml--1)1917 1062545±691.81±0.2245.16±3.433.75±0.710.42±0.08718±634618±2960.82±0.090.44±0.032726±822.30±0.3152.47±2.148.56±1.67*1.17±0.25*1098±103*4604±2500.72±0.060.41±0.042544±592.16±0.3655.90±3.465.00±1.680.74±0.28945±1114904±3130.62±0.100.52±0.052680±472.54±0.2155.17±2.967.62±1.451.00±0.151113±2765138±7950.56±0.160.63±0.09**Abbreviations: AUC, area under the curve; HOMA-IR, homeostatic model assessment of insulin resistance. Data are expressed as mean values±s.e. *Po0.05 vsC/C; **Po0.05 vs H/H.Programming of endothelial dysfunctionL Fan et al256International Journal of Obesity (2013) 254--262& 2013 Macmillan Publishers Limited

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chain reaction. As shown in Supplementary Figure S2a, the levelof eNOS mRNA in the intact abdominal aorta was not differentbetween groups. However, it was positively correlated with Rmax forH/H group of animals (r¼0.72, Po0.05; Supplementary Figure S2b).Intima thicknessDuring the development of atherosclerosis, more connectivetissues build up in intima of the vessel. In the current study, themean intima thickness of the abdominal aorta for H/H animals wasnearly 90% higher than that for C/C (Po0.01; Figure 2a),accompanied by the predominant proliferation of smooth musclecellsdeposited underneath(Figure 2b). Abnormal increase in the intima wall was alsoobserved in diet-switched H/C and C/H animals (Po0.05;Figure 2a).theendothelium monolayerPro-inflammationOn H&E slides, vacuole-like cells were readily apparent, depositingunderneath the endothelium in abdominal aorta rings specificallyin H/H juveniles. However, inflammation with obvious histiocyticinfiltration could not yet be detected at this stage (Figure 3).Nonetheless, proinflammatory markers such as VEGF, TNFa andICAM-1 in aortas from H/H animals were increased in comparisonto C/C offspring (Po0.05, Po0.01). Postweaning diet reversal (H/C)did not significantly reverse the expression of VEGF or TNFa(Figure 4a). However, ICAM-1 tends to be lower in the H/C animalscompared with H/H, but it was not significantly different fromeither the C/C or H/H group. The C/H animals displayed a modest,but significant increase in the expression of VEGF, but had normalexpression levels for TNFa and ICAM-1 (Figure 4a).There was no significant difference in the expression of IL-6,VCAM-1 and MCP-1 between any of these groups (Figure 4b).Furthermore, none of these cytokines was differently expressed inthe renal artery from any group (Supplementary Figure S3).Fibrinolytic systemEndothelial dysfunction usually occurs in conjunction withimpaired fibrinolysic function, predisposing the vessel to aprecoagulant state.36,37In the current study, we observed anincreased expression of PAI-1 in abdominal aorta tissue from H/Hjuveniles in comparison with C/C (Po0.01), along with a notabledecrease in the ratio of t-PA relative to PAI-1 (Po0.01), indicating adisrupting balance between fibrinolysis and coagulation of thevessel (Figure 5). No significant difference in PAI-1 expression wasdetected for both H/C and C/H groups compared with eitherC/C or H/H; however, the t-PA/PAI-1 ratio was significantly higherin the H/C and C/H groups compared with the H/H, but was notdifferent from the C/C group. No notable differences weredetected in renal arteries (Supplementary Figure S4).Of note, there were no sex differences regarding themanifestation of vascular function.DISCUSSIONAmong all the animal models, NHP have the greatest similarity tohumans with obesity and/or diabetes in terms of the alterations inplasma glucose level and pathologic features. It is also the closestanimal model for investigating the complications of metabolicdiseases, such as cardiovascular disorders. In our NHP model,vascular function of the offspring was impaired due to maternalHFD exposure, manifested as depressed endothelium-dependentvasodilatation, thickened intima wall, as well as the developmentof inflammation and prothrombosis. Such manifestation increasesthe susceptibility of the offspring to an increased risk of early-onset atherogenesis. Importantly, the vascular function impair-ments observed in HFD offspring were before the developmentof obesity. Based on our findings, it is clear that both early-programming events and postweaning diet contribute tothe homeostasis of endothelial function. It is equally clear thatthe detrimental effect on endothelial function due to maternalHFD consumption can be partially reversed by a healthy dietintervention.Maternal and early postnatal HFD exposure gives rise to anoffspring phenotype of endothelial dysfunctionIt is recognized that endothelium-dependent vasodilatation is akey measurement of endothelial function. Commonly, impairedarteries have a decreased capacity to dilate fully in response tocertain physiological/pharmacological stimuli. Our findings onthe NHP model showed that the abdominal aorta rings taken fromH/H juveniles lost nearly 50% of dilation capacity, with a potencythat was more than threefold lower in response to theendothelium-dependent vasodilator ACh. The blunted vasodilata-tion response in the H/H NHP offspring is consistent with previousstudies from obese adult rat offspring born to dams fed a fat-richdiet.13,14However, these earlier studies focused on adult offspringrats that had increased adiposity. Our studies demonstrate thatthe endothelial dysfunction occurs in young lean animals (o3%body fat) with similar body weights. Furthermore, our resultsindicate that while the metabolic phenotype is more severe inmales than in females (unpublished observations), there was nosexual dimorphism in the abnormalities of the vascular function.Interestingly, the vasodilation of the renal artery rings taken fromthe same H/H juveniles did not present similar diminishedvasodilatation as those in abdominal aorta rings. Although the56789ACh (-Log M)100255075100C/CH/ HH/CC/HΔΔΔ**Relaxation (%)-10-8-605001000150020002500r = 0.534p = 0.033EC50IAUCC/C H/H H/C C/H0246Contraction force (g)C/C H/H H/C C/H-9.0-8.5-8.0-7.5-7.0**ΔΔEC50 (-Log M)Figure 1.sponse in abdominal aorta tissue. (a) Cumulative concentration-relaxation (%) curves for ACh (?10 to ?5.5 LogM) in abdominalaorta rings against U46619 (9,11-dideoxy-11a, 9a-epoxy-methano-prostaglandin F2a). (b) Corresponding EC50value for each curve.(c) Correlative relationship between EC50and IAUC for juveniles bornto HFD consumption dams. (d) Contracting forces against U46619.Values were expressed as mean±s.e., n¼6--10. **Po0.01 vs C/C,ACh-induced endothelium-dependent vasorelaxation re-DPo0.05 andDDPo0.01 vs H/H.Programming of endothelial dysfunctionL Fan et al257International Journal of Obesity (2013) 254--262& 2013 Macmillan Publishers Limited

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present study could not clarify whether the discrepancy of thevasorelaxation response of these two different arteries was relatedto the difference in the vascular structure and function, the resultsof this study reflect a different pathophysiological alteration inendothelial function related to insulin resistance. While it has beenshown that insulin resistance is related more closely to macro-vasculature than micro-vasculature,38our findings demonstrate avessel-specific dysfunction difference in macro vessels.There are several possibilities that could contribute toendothelial dysfunction in the aorta, including decreased expres-sion of eNOS, an increased diffusion barrier with impairedendothelial signal transduction caused by increased intimathickness, lower bioactivity of eNOS-derived nitric oxide (NO) byreactive oxygen species, reduced availability of L-arginine, orreduced response of vascular smooth muscle to NO.39Amongthese, the last two possibilities are less likely, as the endothelialcells contain concentrations of L-arginine hundreds of timesgreater than those required for the activity of eNOS to synthesizeNO (micromolar range).40,41Abnormalities in vasomotion of thevascular smooth muscle are less likely to develop until theadvanced stage of atherosclerosis.42Thus, the likely majorcontributors are depressed eNOS expression, thickened intimaor decreased bioactivity of NO. In our current studies, we observedno differences in eNOS expression in the H/H aorta, suggestingthat the overall endothelial dysfunction in the aorta of theseanimals was not significantly affected by the expression level ofeNOS. Nonetheless, within the H/H group, the maximal relaxantcapacity of the abdominal aorta rings was positively correlatedwith the eNOS expression level, indicating that eNOS may have aminor contribution to endothelial dysfunction in our model.Thickened intima, commonly presented during the process ofatherogenesis, increases the diffusion barrier of the endothelium-derived factors from endothelial cells to the smooth muscle cellsunderneath and consequently impairs signal transduction in thevasculature. Therefore, the outcome of the greatly increasedintima thickness observed in the abdominal aorta vascular ringcan be a contributor to the notable depression in vasodilatationcapacity found in juvenile H/H offspring. The decreased bioactivityC/C H/H H/C C/H0102030****Intima thickness (?M)10 X NA0.520 X NA0.8C/CH/HFigure 2.juvenile offspring. (b) Immunohistochemistry pictures immunostained with endothelial marker CD31, typical showing the intima thickness forabdominal aorta taken from C/C and H/H animals. Arrows indicate areas of intima thickening, and the top boxes indicate areas of enlargedpictures. Values were expressed as mean±s.e., n¼3--5. *Po0.05 and **Po0.01 vs C/C.Intima thickness of the abdominal aorta tissue. (a) Mean intima thickness value of abdominal aorta tissue for various groups ofProgramming of endothelial dysfunctionL Fan et al258International Journal of Obesity (2013) 254--262& 2013 Macmillan Publishers Limited

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of NO, which contributes to endothelial impairment, could becaused by increased oxidative stress due to excess generation ofreactive oxygen and nitrogen species, such as superoxide anionand peroxynitrite. The increase in reactive oxygen species mightcome from elevated glucose that causes more superoxidegeneration, combined with the low oxygen diffusion associatedwith increased wall thickness. The lower bioactivity of NO weakensits anti-proliferative and anti-inflammatory properties.43,44In thepresent study, we observed an obvious proliferation of smoothmuscle cells deposited underneath the endothelium, which hintsat impairment in the bioactivity of NO. In addition, even thoughdefinite inflammation with histiocytic infiltration under theendothelium was not observed, elevated expression levels ofproinflammatory factors, such as VEGF, TNFa and ICAM-1 weredetected in aorta tissue from H/H juveniles. Highly expressedproinflammatory cytokines and chemokines usually occur at theearly stage of inflammation in the vessel.45,46Vascular inflamma-tion has a pivotal role in all stages of atherosclerosis, frominitiation through progression, and, ultimately, to the occurrenceof thrombotic complications.47,48Atherosclerosis is also thusregarded as a dynamic and progressive pathophysiologicalprocess arising from a combination of inflammation andendothelial dysfunction, both of which were observed in theaorta of our H/H offspring.Decreased bioactivity of NO not only undermines its propertiesof anti-inflammation and anti-proliferation, it also weakens itsability to prevent the development of coagulation.43A vascularpro-thrombotic state is commonly manifested as destroyedfibrinolytic function. Fibrinolysis is the process wherein the bloodclots produced by coagulation are broken down. It is usuallyinitiated by the activation of plasmin, which could be activated inresponse to t-PA, whereas t-PA itself could be inhibited by PAI. Asobserved in our research, excess levels of PAI-1 released from theimpaired endothelium could inhibit the activation of plasminand thereby decrease the break-down of the fibrin mass. Thisultimately destroys the balance between fibrinolysis and coagula-tion in the vessel, thereby promoting atherogenesis. For H/Hjuveniles, the up-regulated expression of PAI-1, together withnotably depressed ratio of t-PA/PAI-1, indicated impaired fibrino-lytic function of the abdominal aorta. These findings are consistentwith other studies that show the coexistence of hypercoagulabilitywith insulin resistance.36,37Although insulin resistance links obesity to generalized meta-bolic disorders, and endothelial dysfunction is the main feature ofboth metabolic disorders and CVD, it is disputed whetherthe occurrence of insulin resistance promotes the induction ofendothelial dysfunction or whether abnormal endothelial functionexacerbates insulin resistance.49--51In any case, insulin resistanceis unlikely to promote atherogenesis independently of other riskfactors. More evidence is required to uncover the role of insulinresistance in endothelial dysfunction. However, our data clearlyshow that endothelial dysfunction and insulin resistance werecoexistent, as demonstrated by impaired endothelium functionconcurrent with elevated fasting insulin levels, IAUC and HOMA-IR.Moreover, a positive correlation between EC50of ACh-inducedvasodilatation and IAUC was observed in the HFD offspring.Postweaning HFD exposure moderately elevates the susceptibilityto abnormalities in vascular endothelial functionIn order to understand whether the high-fat dietary intake itself, orwhether early programming events contribute to the abnormal20 X NA0.810 X NA0.5C/CH/HFigure 3.manifestation for the abdominal aorta rings taken from animals from C/C and H/H. Top boxes indicate areas of enlarged pictures below.Morphological assessment of the abdominal aorta tissue. H&E staining pictures typically showing the different morphologicalProgramming of endothelial dysfunctionL Fan et al259International Journal of Obesity (2013) 254--262& 2013 Macmillan Publishers Limited

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endothelial function in H/H animals, we observed the endothelialfunction in offspring with HFD ingestion limited to the postwean-ing period (C/H). It is interesting that despite a similar metabolicphenotype in H/H and C/H animals, abnormal vasodilatation inthe aorta was only observed in H/H offspring. However, there isevidence of emerging vascular dysfunction as the intima of thevessel of C/H offspring was thickened, and the cytokine VEGFexpression was elevated as compared with C/C. In view of thesefindings, the endothelial dysfunction demonstrated in the youngH/H offspring is primarily in response to early programmingevents, rather than simply an effect of current diet. However, it ishighly likely that continued HFD exposure in the C/H group wouldlikely eventually result in more extensive endothelial dysfunction.Diet intervention after weaning partially improves endothelialdysfunctionIf maternal HFD consumption during pregnancy and earlypostnatal period resulted in endothelial dysfunction in theoffspring, then it is important to know whether the damage isreversible. In order to address this critical question, some of ourHFD offspring underwent a diet switch to a normal chow afterweaning (H/C). Importantly, there was no obvious difference inACh-induced vasorelaxation curve detected in aorta rings inH/C juveniles compared with C/C, suggesting at least someimprovement was produced in the diet intervention group. In fact,the vessels of these H/C animals displayed higher sensitivity inresponse to ACh. This was surprising and the underlying causesof this hypersensitivity are unknown. However, there was stillevidence that increased intima wall and elevated pro-inflamma-tion cytokine expression persisted in the H/C animals, suggestingthat the diet intervention was not sufficient to completelynormalize the damage caused by early HFD exposure. A numberof causes could explain these findings: they could be the resultof the relatively short time period of diet intervention, or as aconsequence of the programming events that have alreadyaltered certain genotypes, or both. The relatively normalmanifestation of the metabolic parameters, such as body weight,VEGF mRNA / 18sC/C H/H H/C C/HC/C H/H H/C C/HC/C H/H H/C C/H0123****TNF? mRNA / 18s0.00.51.01.52.00.00.51.01.52.0**ICAM-1 mRNA / 18s*MCP-1 mRNA / 18S01234IL-6 mRNA / 18sC/C H/H H/C C/H0.000.080.160.240.32VCAM-1 mRNA / 18s0.00.51.01.52.0C/C H/H H/C C/H C/C H/H H/C C/HFigure 4.abdominal aorta tissue of Juvenile offspring. (b) Relative expression levels of IL-6, VCAM-1 and MCP-1 in abdominal aorta tissue of Juvenileoffspring. Values were expressed as mean±s.e., n¼6--17. *Po0.05 and **Po0.01 vs C/C.Expression of proinflammatory factors in abdominal aorta tissue. (a) Relative expression levels of VEGF, TNFa and ICAM-1, inPAI-1 mRNA / 18sC/C H/H H/C C/H C/C H/H H/C C/H012**t-PA / PAI-102468*ΔΔΔt-PA / PAI-1Figure 5.(a) Relative expression level of PAI-1 and (b) the ratio between t-PAand PAI-1 in abdominal aorta tissue of juvenile offspring. Valueswere expressed as mean±s.e., n¼6--17. *Po0.05, **Po0.01 vs C/CandDPo0.05,DDPo0.01 vs H/H.Expression of fibrinolytic factors in abdominal aorta tissue.Programming of endothelial dysfunctionL Fan et al260International Journal of Obesity (2013) 254--262& 2013 Macmillan Publishers Limited